It takes two to tango without O2: Metabolic interactions between breviate protists and bacteria in anoxia

Breviates are anaerobic protists and among the closest unicellular relatives of animals and fungi, yet their ecology and metabolism remain poorly understood. In this thesis, I investigated the distribution, metabolic potential, and bacterial interactions of breviates in low-oxygen environments. Breviates rely on bacteria both as a food source and potentially as metabolic partners. Breviates are predicted to produce hydrogen gas via FeFe-hydrogenases, whose activity requires low hydrogen partial pressure; therefore, hydrogen-consuming partners are essential to sustain breviate metabolism. Using metagenomics, 16 high-quality bacterial genomes were reconstructed from protist-associated Arcobacteraceae, Desulfovibrionaceae, and Terasakiellaceae, revealing one new genus and nine new species. All bacterial genomes encoded NiFe-hydrogenases, indicating hydrogen uptake potential, long with diverse sulfur and nitrogen metabolic pathways. The relative abundance of associated bacteria shifted with the availability of electron acceptors, with nitrate reducers being more abundant in the presence of nitrate and sulfate reducers in the presence of sulfate. Growth experiments showed that breviates grew well in the presence of nitrate when hydrogenotrophic denitrifying bacteria were present, as shown before for the breviate Lenisia limosa. In addition, growth experiments showed that Pygsuia biforma reached significantly higher cell densities when co-cultured with a sulfate-reducing bacterium (SRB) under sulfate-replete conditions than under sulfate-free conditions or without SRB. Transcriptomic analyses revealed that sulfate deprivation induced oxidative stress responses in P. biforma, whereas sulfate availability supported a more active metabolic state. Together, these results support a syntrophic model in which SRB perform dissimilatory sulfate reduction using breviate-derived hydrogen as an electron donor, while the breviates use the resulting sulfide for Fe–S cluster and amino acid biosynthesis. This work advances our understanding of how breviate protists interact with bacteria in low-oxygen environments and how these interactions influence the hydrogen, nitrogen, and sulfur cycles